Immunomodulatory Effects of BRAF, MEK, and CDK4/6 Inhibitors: Implications for Combining Targeted Therapy and Immune Checkpoint Blockade for the Treatment of Melanoma

Abstract

The recent advent of targeted and immune-based therapies has revolutionized the treatment of melanoma and transformed outcomes for patients with metastatic disease. The majority of patients develop resistance to the current standard-of-care targeted therapy, dual BRAF and MEK inhibition, prompting evaluation of a new combination incorporating a CDK4/6 inhibitor. Based on promising preclinical data, combined BRAF, MEK and CDK4/6 inhibition has recently entered clinical trials for the treatment of BRAF^V600 melanoma. Interestingly, while BRAF- and MEK-targeted therapy was initially developed on the basis of potent tumor-intrinsic effects, it was later discovered to have significant immune-potentiating activity. Recent studies have also identified immune-related impacts of CDK4/6 inhibition, though these are less well defined and can be both immune-potentiating and immune-inhibitory. BRAF^V600 melanoma patients are also eligible to receive immunotherapy, specifically checkpoint inhibitors against PD-1 and CTLA-4. The immunomodulatory activity of BRAF/MEK-targeted therapies has prompted interest in combination therapies incorporating these with immune checkpoint inhibitors, however recent clinical trials investigating this approach have produced variable results. Here, we summarize the immunomodulatory effects of BRAF, MEK and CDK4/6 inhibitors, shedding light on the prospective utility of this combination alone and in conjunction with immune checkpoint blockade. Understanding the mechanisms that underpin the clinical efficacy of these available therapies is a critical step forward in optimizing novel combination and scheduling approaches to combat melanoma and improve patient outcomes.

Keywords: BRAF/MEK inhibition; CDK4/6 inhibition; immune checkpoint blockade; immunotherapy; melanoma; targeted therapeutic drugs.

Figure 1

MAPK/ERK and p16/CyclinD-CDK4/6/RB signaling pathways in health and disease. (A) In healthy cells, growth factors bind receptor tyrosine kinases on the surface of the cell to stimulate proliferation and survival through the MAPK/ERK signaling pathway, which is mediated through a cascade of phosphorylation events (NRAS → BRAF → MEK → ERK). MAPK/ERK signaling promotes cyclin D1-CDK4/6 complex formation. Inhibition of CDK4/6 by p16 prevents CDK4/6-mediated phosphorylation of RB, thereby allowing RB to carry out its tumor suppressive function. (B) BRAF^V600 mutations and loss of p16 are often seen in melanoma. BRAF^V600 is constitutively active and promotes aberrant overactivation of MAPK/ERK signaling. In the absence of p16, CDK4/6 phosphorylates and inhibits the tumor suppressor RB. These events lead to uncontrolled cellular proliferation and survival. (C) Clinical inhibitors against BRAF^V600, MEK and CDK4/6 block MAPK/ERK signaling and CDK4/6-mediated suppression of RB, thereby attenuating cell cycle and survival.

Figure 2

Immunomodulation by oncogenic BRAF and inhibitors of BRAF and MEK in melanoma. (A) BRAF^V600 promotes tumor progression through tumor-intrinsic activation of MAPK/ERK signaling and increased production of immunosuppressive cytokines. MAPK/ERK signaling via oncogenic BRAF^V600 is associated with increased frequencies of Tregs and MDSCs in the tumor microenvironment. In T cells, RAF is not overactive and there is no aberrant MAPK/ERK signaling. (B) BRAF and MEK inhibition blocks MAPK/ERK signaling in BRAF^V600 tumor cells and induces immunogenic cell death. Inhibition of MAPK/ERK promotes the upregulation of MHC I and expression of melanoma associated antigens by tumor cells and is associated with increased frequencies of T cells and pro-inflammatory cytokines in the tumor microenvironment. BRAF inhibition is also associated with a reduction in intratumoral immunosuppressive cells including Tregs and MDSCs. In T cells and other cells with wild type BRAF, BRAF inhibition promotes paradoxical MAPK/ERK activation through RAF dimerization and dimer-dependent enzyme transactivation, enhancing the proliferation and survival of these cells. MDSC, myeloid derived suppressor cell; Treg, Regulatory T cell.

Figure 3

Immunomodulatory effects of CDK4/6 inhibition. (A) CDK4/6 inhibition modulates anti-tumor immunity through multiple mechanisms. In tumors cells, CDK4/6 inhibition leads to hypophosphorylated RB, which binds to and inhibits the activity of E2F transcription factors. Reduced E2F activity leads to an induction of Type III interferons (IFN), resulting in paracrine IFN signaling and upregulation of MHC I. This induction of IFN is due to the suppression DNMT1, (an E2F target gene), which reduces methylation of endogenous retroviral (ERV) genes, thereby promoting their expression and inducing viral mimicry. Hypophosphorylated RB also promotes activation of NFκB and subsequent upregulation of T cell chemoattractants and PD-L1. (B) CDK4/6 inhibition also prevents PD-L1 degradation, further enhancing PD-L1 protein expression on the cells surface. In T cells, NFAT activity is restrained by CDK4/6-mediated phosphorylation. Following CDK4/6 inhibition, hypophosphorylated NFAT translocates to the nucleus and upregulates expression of effector genes. Compared to other lymphocyte populations, T regulatory cells appear particularly susceptible to the anti-proliferative effects of CDK4/6 inhibition.